Insulin-like growth factor II antisense oligonucleotide sequences and methods of using same to modulate cell growth

ABSTRACT

This invention relates to oligonucleotides complementary to the IGF-II genes which modulate tumor cell growth in mammals This invention is also related to methods of using such compounds in inhibiting the growth of tumor cells in mammals This invention also relates to pharmaceutical compositions comprising a pharmaceutically acceptable excipient and an effective amount of a compound of this invention.

REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional ApplicationSerial No. 60/082,791 filed Apr. 23, 1998, which application isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] Field of the Invention

[0003] This invention relates to oligonucleotides that are complementaryto mammalian insulin-like growth factor II (IGF II) genes whicholigonucleotides modulate tumor cell growth in mammals. This inventionis also related to methods of using such compounds in inhibiting thegrowth of tumor cells in mammals. This invention also relates topharmaceutical compositions comprising a pharmaceutically acceptableexcipient and an effective amount of a compound of this invention.

REFERENCES

[0004] The following publications, patent applications and patents arecited in this application:

[0005] 1. Toretsky, J. A. and Helman, L. J. Involvement of IGF-II inhuman cancer, J Endocrinol. 149: 367-72, 1996.

[0006] 2. Werner, H. and LeRoith, D. The role of the insulin-like growthfactor system in human cancer, Adv Cancer Res. 68: 183-223, 1996.

[0007] 3. Rogler, C. E., Yang, D., Rossetti, L., Donohoe, J., Alt, E.,Chang, C. J., Rosenfeld, R., Neely, K., and Hintz, R. Altered bodycomposition and increased frequency of diverse malignancies ininsulin-like growth factor-II transgenic mice, J Biol Chem. 269:13779-84, 1994.

[0008] 4. Bates, P., Fisher, R., Ward, A., Richardson, L., Hill, D. J.,and Graham, C. F. Mammary cancer in transgenic mice expressinginsulin-like growth factor II (IGF-II) [see comments], Br J Cancer. 72:1189-93, 1995.

[0009] 5. Cullen, K. J., Lippman, M. E., Chow, D., Hill, S., Rosen, N.,and Zwiebel, J. A. Insulin-like growth factor-II overexpression in MCF-7cells induces phenotypic changes associated with malignant progression,Mol Endocrinol. 6: 91-100, 1992.

[0010] 6. Werner, H., Adamo, M., Roberts, C. T., Jr., and LeRoith, D.Molecular and cellular aspects of insulin-like growth factor action,Vitam Horm. 48: 1-58, 1994.

[0011] 7. Curcio, L. D., Bouffard, D. Y., and Scanlon, K. J.Oligonucleotides as modulators of cancer gene expression, PharmacolTher. 74: 317-32, 1997.

[0012] 8. Narayanan, R. and Akhtar, S. Antisense therapy, Curr OpinOncol. 8: 509-15, 1996.

[0013] 9. Ho, P. T. and Parkinson, D. R. Antisense oligonucleotides astherapeutics for malignant diseases, Semin Oncol. 24: 187-202, 1997.

[0014] 10. Crooke, S. T. and Bennett, C. F. Progress in antisenseoligonucleotide therapeutics, Annu Rev Pharmacol Toxicol. 36: 107-29,1996.

[0015] 11. Christofori, G., Naik, P., and Hanahan, D. A second signalsupplied by insulin-like growth factor II in oncogene- inducedtumorigenesis, Nature. 369: 414-8, 1994.

[0016] 12. El-Badry, O. M., Minniti, C., Kohn, E. C., Houghton, P. J.,Daughaday, W. H., and Helman, L. J. Insulin-like growth factor II actsas an autocrine growth and motility factor in human rhabdomyosarcomatumors, Cell Growth Differ. 1: 325-31, 1990.

[0017] 13. Kim, K. W., Bae, S. K., Lee, O. H., Bae, M. H., Lee, M. J.,and Park, B. C. Insulin-like growth factor II induced by hypoxia maycontribute to angiogenesis of human hepatocellular carcinoma, CancerRes. 58: 348-51, 1998.

[0018] 14. Volpert, O., Jackson, D., Bouck, N., and Linzer, D. I. Theinsulin-like growth factor II/mannose 6-phosphate receptor is requiredfor proliferin-induced angiogenesis, Endocrinology. 137: 3871-6, 1996.

[0019] 15. Lin, S. B., Hsieh, S. H., Hsu, H. L., Lai, M. Y., Kan, L. S.,and Au, L. C. Antisense oligodeoxynucleotides of IGF-II selectivelyinhibit growth of human hepatoma cells overproducing IGF-II, J Biochem(Tokyo). 122: 717-22, 1997.

[0020] 16. Steller, M. A., Delgado, C. H., Bartels, C. J., Woodworth, C.D., and Zou, Z. Overexpression of the insulin-like growth factor-1receptor and autocrine stimulation in human cervical cancer cells,Cancer Res. 56: 1761-5, 1996.

[0021] 17. Steller, M. A., Delgado, C. H., and Zou, Z. Insulin-likegrowth factor II mediates epidermal growth factor-induced mitogenesis incervical cancer cells, Proc Natl Acad Sci U S A. 92: 11970-4, 1995.

[0022] 18. Choy et al., “Molecular mechanisms of drug resistanceinvolving ribonucleotide reductase: hydroxyurea resistance in a seriesof clonally related mouse cell lines selected in the presence ofincreasing drug concentrations” Cancer Res. 48:2029-2035 (1988)

[0023] 19. Fan et al., “Ribonucleotide reductase R2 component is a novelmalignancy determinant that cooperates with activated oncogenes todetermine transformation and malignant potential” Proc. Natl. Acad. SciUSA 93:14036-40 (1996)

[0024] 20. Huang and Wright, “Fibroblast growth factor mediatedalterations in drug resistance and evidence of gene amplification”Oncogene 9:491-499 (1994)

[0025] 21. Uhlmann et al. Chem Rev. 90:534-583 (1990)

[0026] 22. Agrawal et al. Trends Biotechnol. 10:152-158 (1992)

[0027] 23. Remington's Pharmaceutical Sciences, Mace Publishing Company,Philadelphia Pa. 17^(th) ed. (1985)

[0028] 24. Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, New York (1989, 1992)

[0029] 25. Ausubel et al., Current Protocols in Molecular Biology, JohnWiley and Sons, Baltimore Maryland (1989)

[0030] 26. Perbal, A Practical Guide to Molecular Cloning, John Wiley &Sons, New York (1988)

[0031] 27. Hurta and Wright, “Malignant transformation by H-ras resultsin aberrant regulation of ribonucleotide reductase gene expression bytransforming growth factor-beta” J. Cell Biochem 57:543-556 (1995)

[0032] 28. Dreeley et al., Science, 258:1650-1654 (1992)

[0033] 29. Nielsen et al.; Science (1991) 354:1497

[0034]30. Good and Nielsen; “Inhibition of translation and bacterialgrowth by peptide nucleic acid targeted to ribosomal RNA”, PNAS USA(1998) 95:2073-2076

[0035] 31. Buchardt, deceased, et al., U.S. Pat. No. 5,766,855

[0036] 32. Buchardt, deceased, et al., U.S. Pat. No. 5,719,262

[0037] 33. U.S. Pat. No. 5,034,506

[0038] 34. Altschul, et al. “Basic local alignment search tool”, J. Mol.Biol. (1990) 215:403-10;

[0039] 35. Devereux J. et al., “A comprehensive set of sequence analysisprograms for the VAX”, Nucleic Acids Res. (1984) 12:387-395;

[0040] 36. Chang et al.; Somatic Gene Therapy, CRC Press, Ann ArborMich. (1995);

[0041] 37. Vega et al.; Gene Targeting, CRC Press, Ann Arbor Mich.(1995)

[0042] 38. Vectors: A Survey of Molecular Cloning Vectors and TheirUses, Butterworths, Boston Mass. (1988)

[0043] 39. Sullivan, U.S. Pat. No. 5,225,347

[0044] 40. U.S. Pat. No. 5,023,252, issued Jun. 11, 1991

[0045] 41. Feigner et al., U.S. Pat. No. 5,580,859

[0046] All of the above publications, patent applications and patentsare herein incorporated by reference in their entirety to the sameextent as if each individual publication, patent application or patentwas specifically and individually indicated to be incorporated byreference in its entirety.

[0047] State of the Art

[0048] Insulin-like growth factor II (IGF-II) is a 67 amino acidpolypeptide growth factor that is widely expressed in the developinghuman embryonic tissues and is related to the growth and differentiationof various tissues. After birth, the expression is progressivelyextinguished in almost all human tissues. In adult humans, serum levelsof approximately 100 ng/ml are mainly produced by the liver. Thebiological functions of IGF-II are mediated through its binding toeither the IGF-II receptor (related to carbohydrate metabolism, motilityof malignant cells and/or tumor-induced angiogenesis) or the IGF-Ireceptor (related to signal transduction pathway and mitogenesis).

[0049] IGF-II has been implicated in tumor progression and metastasis bya variety of mechanisms in many tumors (reviewed in (1, 2)). Tumors withextensive involvement of IGF-II include childhood tumors such asrhabdomyosarcoma, Wilms' tumor and neuroblastoma. These tumorsdemonstrate overexpression of IGF-II, show existence of a paracrine orautocrine loop and result in inhibition of tumor growth or metastasisupon blockage of the loop. IGF-II contributes to tumor growth andmetastasis to varying degrees in a variety of tumors includingosteosarcoma, breast carcinoma, hepatoblastoma, germ cell tumors,hepatocellular carcinoma, adrenocortical carcinoma, lung tumors,leiomyosarcoma, brain tumors and colon carcinoma. Furthermore, thedirect role of IGF-II in oncogenesis has been elucidated by transgenicmice and human cell lines overexpressing it (3-5).

[0050] The human IGF-II gene is located on chromosome 11p15 justdownstream of insulin gene and spans 30 kb (reviewed in (6);see FIG. 1).It consists of 9 exons of which exons 7, 8 and part of 9 encode aprecursor protein. Exons 1, 4, 5, and 6 are each preceded by distinctpromoters P1, P2, P3 and P4. Promoter P1 is active only in adult liver,while P2-4 are active in most fetal tissues. There are a few adulttissues that express low amount of transcripts from P2, 3 and 4 (fetaltranscripts). Four major mRNA species (6 Kb, 4.8-5 kb and 2.2 kb forfetal transcripts and 5.3 kb for adult transcript) have been identifiedwhich are generated from distinct promoters and by differentialsplicing. It appears that overexpression of IGF-II observed in variousprimary cancers and cell lines results from reactivation (in liver) oroverexpression (in other organs) of fetal mRNA species whose expressionis mainly derived from P3 and P4. These fetal transcripts contain unique5′ untranslated regions (5′UTR containing exons 4 or 5 or 6) that areabsent in the adult transcript derived from P1(5′UTR containing exons 1,2 and 3).

[0051] Antisense oligonucleotides (AS-ODNs) have been widely utilized toinhibit gene expression in a target-specific manner by sequence-specifichybridization to target mRNA. In numerous studies, antisenseoligonucleotide-mediated repression of oncogenes has revealed that thesecompounds are not only extremely useful for delineating biochemicalmechanisms governing oncogenesis (7), but also considerably promising asnovel therapeutic compounds for the treatment of human cancer (8, 9). Inaddition, relatively less toxicity has been attributed tooligonucleotide-based therapeutics (10).

[0052] A few studies (11, 15-17) have shown that certain antisenseoligonucleotides targeted against human or mouse adult IGF-IItranscripts were effective in interfering with tumor cell proliferationin vitro. In one study (15), the suppression of IGF-II production by anantisense oligonucleotide targeting the translation start site of humanadult transcript has resulted in growth inhibition of humanhepatocellular carcinoma cell lines, HuH-7 and HepG2. In another studies(16,17) utilizing human cervical cancer cell line, an antisenseoligonucleotide targeting the protein coding region of IGF-II was shownto inhibit epidermal growth factor (EGF)-induced mitogenic effect.

[0053] Therefore, it would be desirable to identify antisenseoligonucleotides directed against IGF-II which act to inhibit theexpression and production of IGF-II with higher specificity and withless toxicity.

SUMMARY OF THE INVENTION

[0054] This invention is directed to antisense oligonucleotides whichmodulate the expression of the IGF-II genes and production of IGF-II inmammals and pharmaceutical compositions comprising such antisenseoligonucleotides. This invention is also related to methods of usingsuch antisense oligonucleotides for inhibiting tumor growth andmetastasis in mammals.

[0055] Accordingly, in one of its composition aspects, this invention isdirected to an antisense oligonucleotide, which oligonucleotide fromabout 3 to about 100 nucleotides comprising nucleotides complementary tothe mammalian fetal IGF-II mRNA. The antisense oligonucleotide may benuclease resistant and may have one or more phosphorothioateinternucleotide linkages. The antisense oligonucleotide may furthercomprise additional nucleotides which are not complementary to theIGF-II mRNA. The oligonucleotides may comprise a sequence selected fromgroup consisting of SEQ ID NOs:1 to 15 from Table 1.

[0056] This invention is also directed to an antisense oligonucleotide,which oligonucleotide from about 20 to about 100 nucleotides comprisingnucleotides complementary to the mammalian adult IGF-II mRNA selectedfrom the group consisting of SEQ ID NOs:17-31 from Table 2.

[0057] In another of its composition aspects, this invention is directedto a vector comprising an antisense oligonucleotide sequence from about3 to 100 nucleotides comprising a sequence complementary to the 5′untranslated region of mammalian fetal IGF-II mRNA.

[0058] In another of its composition aspects, this invention is directedto a vector comprising an antisense oligonucleotide sequence from about20 to 100 nucleotides comprising a sequence selected from the groupconsisting of SEQ ID NOs:1 7-31 in Table 2.

[0059] In still another of its composition aspects, this invention isdirected to a pharmaceutical composition comprising a pharmaceuticallyacceptable excipient and an effective amount of an antisenseoligonucleotide from about 3 to about 100 nucleotides comprisingnucleotides complementary to the mammalian fetal IGF-II mRNA. Theoligonucleotides may comprise a sequence selected from group consistingof SEQ ID NOs:1 to 15 from Table 1.

[0060] In still another of its composition aspects, this inveniton isdirected to a pharmaceutical composition comprising a pharmaceuticallyacceptable excipient and an effective amount of an antisenseoligonucleotide from about 20 to about 100 nucleotides comprising asequence selected from the group consisting of SEQ ID NOs:17-31 fromTable 2.

[0061] In one of its method aspects, this invention is directed to amethod for inhibiting the growth of a mammalian tumor comprising,administering to a mammal suspected of having the tumor an effectiveamount of an antisense oligonucleotide from about 3 nucleotides to about100 nucleotides complementary to mammalian fetal IGF-II mRNA underconditions such that the growth of the tumor is inhibited. The antisenseoligonucleotide may be administered with a chemotherapeutic agent. Theoligonucleotide may comprise a sequence selected from group consistingof SEQ ID NOs:1 to 15 from Table 1.

[0062] This invention is also directed to a method for inhibiting thegrowth of a mammalian tumor comprising, administering to a mammalsuspected of having the tumor an effective amount of an antisenseoligonucleotide from about 20 nucleotides to about 100 nucleotidescomplementary to mammalian adult IGF-II mRNA selected from the groupconsisting of SEQ ID NOs:17-31 from Table 2 under conditions such thatthe growth of the tumor is inhibited.

[0063] In another of its method aspects, this invention is directed to amethod for inhibiting the metastasis of a mammalian tumor comprising,administering to a mammal suspected of having a metastatic tumor aneffective amount of an antisense oligonucleotide from about 3nucleotides to about 100 nucleotides complementary to the mammalianfetal IGF-II mRNA under conditions such that the metastasis of the tumoris inhibited. The antisense oligonucleotide may be administered with achemotherapeutic agent. The oligonucleotides may comprise a sequenceselected from group consisting of SEQ ID NOs:1 to 15 from Table 1.

[0064] This invention is also directed to a method for inhibiting themetastasis of a mammalian tumor comprising, administering to a mammalsuspected of having a metastatic tumor an effective amount of anantisense oligonucleotide from about 20 nucleotides to about 100nucleotides complementary to the mammalian adult IGF-II mRNA selectedfrom the group consisting of SEQ ID NOs:1 7-31 from Table 2 underconditions such that the metastasis of the tumor is inhibited.

BRIEF DESCRIPTION OF THE DRAWINGS

[0065]FIG. 1 is map of the human IGF-II gene and alternativelytranscribed and spiced mRNAs. The numbered boxes (1-9) indicate theexons of IGF-II gene. Four promoters (P1-P4) are also indicated witharrows. Various IGF-II mRNA SPecies are depicted in the lower part ofthe figure with their corresponding sizes. The solid boxes representcoding regions of the IGF-II precursor protein.

[0066] FIGS. 2A-D are graphs of the percentage of inhibition of thecolony forming ability of different cell lines by administration of theindicated antisense oligonucleotides.

[0067]FIG. 2A shows the percentage inhibition of the humanrhabdomyosarcoma cell line RD;

[0068]FIG. 2B shows percentage inhibition of the human prostate cancercell line PC-3;

[0069]FIG. 2C shows the percentage inhibition of the human pancreaticcancer cell line AsPC-1:

[0070]FIG. 2D shows the percentage inhibition of the human neuroblastomacell line SK-N-AS.

[0071]FIG. 3 is an autoradiograph of Northern Blots of RNA from eitherhuman neuroblastoma cell line SK-N-AS or rhabdomyosarcoma cell line (RD)after administration with antisense oligonucleotides: GTI4006 [SEQ IDNO:6] or GTI4011 [SEQ ID NO:11]

[0072]FIG. 4 is a photograph of a Western Blot of IGF-II expression inhuman neuroblastoma cells after treatment with different antisenseoligodeoxynucleotides.

[0073]FIG. 5 is a photograph of a Western Blot of IGF-II expression inhuman rhabdomyosarcoma cells after treatment with different antisenseoligodeoxynucleotides.

[0074]FIG. 6A is a graph of the volume of a tumor following injection ofhuman neuroblastoma cells (SK-N-AS) in mice with administration ofvarious antisense oligonucleotides or without (control).

[0075]FIG. 6B is a graph of the weight of a tumor 20 days afterinjection of human neuroblastoma cells (SK-N-AS) in mice withadministration of various antisense oligonucleotides or without(control).

[0076]FIG. 7A is a graph of the volume of a tumor following injection ofhuman Wilms' tumor cells (G401) in mice with administration of variousantisense oligonucleotides or without (control).

[0077]FIG. 7B is a graph of the weight of a tumor 20 days afterinjection of human Wilms' tumor cells (G401) in mice with administrationof various antisense oligonucleotides or without (control).

[0078]FIG. 8 is an autoradiograph of a Northern Blot of IGF-II mRNAlevels in human neuroblastoma (SK-N-AS) tumors following treatment withantisense oligonucleotide GTI4006 [SEQ ID NO:6].

[0079]FIG. 9 is a photograph of a Western blot of IGF-II protein levelsin human neuroblastoma (SK-N-AS) tumors following treatment with variousantisense oligonucleotides. The band below is a photograph of the gelstained with India ink to show the total protein loaded.

[0080]FIG. 10 is a graph of the average number of lung metastases permouse by the human melanoma cell line (C8161) after treatment of thecell line with the various antisense oligonucleotides.

[0081]FIG. 11 is part of the nucleotide sequence of the human IGF-IIgene.

[0082]FIG. 11A is the sequence of exon 4 [SEQ ID NO:34],

[0083]FIG. 11B is the sequence of exon 5 [SEQ ID NO:35],

[0084]FIG. 11C is the sequence of exon 6 [SEQ ID NO:36] and

[0085]FIG. 11D is the sequence of exons 7-9 [SEQ ID NO:37].

DETAILED DESCRIPTION OF THE INVENTION

[0086] This invention relates to oligonucleotides that are complementaryto mammalian IGF II genes which oligonucleotides modulate tumor cellgrowth in mammals. It appears that overexpression of IGF-II observed invarious human primary cancers and cell lines results from reactivation(in liver) or overexpression (in other organs) of fetal mRNA species.Accordingly, antisense oligonucleotides designed to specifically targetfetal transcripts in the 5′UTR, leaving adult transcripts intact, willbe highly specific for targeting tumor cells.

[0087] Without being limited to a theory or mechanism, it is believedthat these antisense compounds will exert their antitumor activity bynot only suppressing autocrine growth of tumor cells and possiblyinducing apoptosis, but also inhibiting autocrine/paracrine function ofIGF-II, such as tumor cell motility and/or induction of endothelial cellmigration and angiogenesis.

[0088] Definitions:

[0089] As used herein, the following terms have the following meanings:

[0090] The term “antisense oligonucleotide” as used herein means anucleotide sequence that is complementary to the desired mRNA. Theantisense oligonucleotide is complementary to any portion of a mammalianIGF-II mRNA that effectively acts as a target for inhibiting IGF-IIexpression. Preferably, the antisense oligonucleotide is complementaryto the 5′ untranslated region of the IGF-II fetal transcript. Morepreferably, the antisense oligonucleotide is complementary to thenucleotide sequence of exons 4, 5 or 6 as set forth in FIGS. 11A-C.

[0091] Without being limited to any theory or mechanism, it is generallybelieved that the activity of antisense oligonucleotides depends on thebinding of the oligonucleotide to the target nucleic acid (e.g. to atleast a portion of a genomic region, gene or mRNA transcript thereof),thus disrupting the function of the target, either by hybridizationarrest or by destruction of target RNA by RNase H (the ability toactivate RNase H when hybridized to RNA).

[0092] The term “oligonucleotide” refers to an oligomer or polymer ofnucleotide or nucleoside monomers consisting of naturally occurringbases, sugars, and inter-sugar (backbone) linkages. The term alsoincludes modified or substituted oligomers comprising non-naturallyoccurring monomers or portions thereof, which function similarly. Suchmodified or substituted oligomers may be preferred over naturallyoccurring forms because of the properties such as enhanced cellularuptake, or increased stability in the presence of nucleases. The termalso includes chimeric oligonucleotides which contain two or morechemically distinct regions. For example, chimeric oligonucleotides maycontain at least one region of modified nucleotides that conferbeneficial properties (e.g. increased nuclease resistance, increaseduptake into cells) or two or more oligonucleotides of the invention maybe joined to form a chimeric oligonucleotide.

[0093] The antisense oligonucleotides of the present invention may beribonucleic or deoxyribonucleic acids and may contain naturallyoccurring or synthetic monomeric bases, including adenine, guanine,cytosine, thymine and uracil. The oligonucleotides may also containmodified bases such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl,2-propyl and other alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-azauracil, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiouracil,8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines,8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines,8-amino guanine, 8-thiol guanine, 8-thioalkyl guanines, 8-hydroxylguanine and other 8-substituted guanines, other aza and deaza uracils,thymidines, cytosines or guanines, 5-trifluoromethyl uracil and5-trifluoro cytosine. The modifications may also include attachment ofother chemical groups such as methyl, ethyl, propyl groups to thevarious parts of the oligonucleotides including the sugar, base orbackbone components.

[0094] The antisense oligonucleotides of the invention may also comprisemodified phosphorus oxygen heteroatoms in the phosphate backbone, shortchain alkyl or cycloalkyl intersugar linkages or short chain heteroatomor heterocyclic intersugar linkages. For example, the antisenseoligonucleotides may contain methyl phosphonates, phosphorothioates,phosphorodithioates, phosphotriesters, and morpholino oligomers. Theantisense oligonucleotides may comprise phosphorothioate bonds linkingbetween the four to six 3′-terminus nucleotides. The phosphorothioatebonds may link all the nucleotides. The phosphorothioate linkages may bemixed R_(P) and S_(P) enantiomers, or they may be stereoregular orsubstantially stereoregular in either R_(P) or S_(P) form.

[0095] The antisense oligonucleotides may also have sugar mimetics. Theoligonucleotide may have at least one nucleotide with a modified baseand/or sugar, such as a 2′-O-substituted ribonucleotide. For purposes ofthe invention, the term 2′-O-substituted” means substitution of the 2′position of the pentose moiety with an -O-lower alkyl group containing1-6 saturated or unsaturated carbon atoms, or with an -O-aryl or allylgroup having 2-6 carbon atoms, wherein such alkyl, aryl or allyl groupmay be unsubstituted or may be substituted, e.g., with halo, hydroxy,trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl,carbalkoxyl, or amino groups. The oligonucleotides of the invention mayinclude four or five ribonucleotides 2′-O-alkylated at their 5′ terminusand/or four or five ribonucleotides 2′-O-alylated at their 3′ terminus.

[0096] The antisense oligonucleotides of the invention may also comprisenucleotide analogues wherein the structure of the nucleotide isfundamentally altered. An example of such an oligonucleotide analogue isa peptide nucleic acid (PNA) wherein the deoxyribose (or ribose)phosphate backbone in DNA (or RNA) is replaced with a polyamide backbonewhich is similar to that found in peptides (Nielsen et al.²⁹; Good andNielsen³⁰; Buchardt, deceased, et al.³¹, U.S. Pat. No. 5,766,855;Buchardt, deceased, et al.³², U.S. Pat. No. 5,719,262). PNA analogueshave been shown to be resistant to degradation by enzymes and to haveextended lives in vivo and in vitro. PNAs also bind more strongly to acomplementary DNA sequence than to a naturally occurring nucleic acidmolecule due to the lack of charge repulsion between the PNA strand andthe DNA strand.

[0097] The oligonucleotides of the present invention may also includeother nucleotides comprising polymer backbones, cyclic backbones, oracyclic backbones. For example, the nucleotides may comprise morpholinobackbone structures (U.S. Pat. No. 5,034,506 (33)).

[0098] The oligonucleotides of the present invention are “nucleaseresistant” when they have either been modified such that they are notsusceptible to degradation by DNA and RNA nucleases or alternativelythey have been placed in a delivery vehicle which in itself protects theoligonucleotide from DNA or RNA nucleases. Nuclease resistantoligonucleotides include, for example, methyl phosphonates,phosphorothioates, phosphorodithioates, phosphotriesters, and morpholinooligomers. Suitable delivery vehicles for conferring nuclease resistanceinclude, for example liposomes.

[0099] The oligonucleotides of the present invention may also containgroups, such as groups for improving the pharmacokinetic properties ofan oligonucleotide, or groups for improving the pharmacodynamicproperties of an oligonucleotide.

[0100] The antisense oligonucleotides are preferably selected from thesequence complementary to the IGF-II gene such that the sequenceexhibits the least likelihood of showing duplex formation, hair-pinformation, and homooligomer/sequence repeats but has a high to moderatepotential to bind to the IGF-II gene sequences. These properties may bedetermined using the computer modeling program OLIGO Primer AnalysisSoftware, Version 5.0 (distributed by National Biosciences, Inc.,Plymouth, Minn.). This computer program allows the determination of aqualitative estimation of these five parameters.

[0101] Alternatively, the antisense oligonucleotides may also beselected on the basis that the sequence is highly conserved for theIGF-II gene between two or more mammalian species. These properties maybe determined using the BLASTN program (Altschul, et al.(34)) of theUniversity of Wisconsin Computer group (GCG) software (Devereux J. etal.(35)) with the National Center for Biotechnology Information (NCBI)databases.

[0102] The antisense oligonucleotides may include mutations, such assubstitutions, insertions and deletions. Preferably there will be lessthat 10% of the sequence having mutations.

[0103] The antisense oligonucleotides generally comprise from at leastabout 3 nucleotides or nucleotide analogs, more preferably they are atleast about 5 nucleotides, more preferably they are at least about 7nucleotides, more preferably they are at least about 9 nucleotides andmost preferably they are at least about 20 nucleotides. The antisenseoligonucleotides are preferably less than about 100 nucleotides ornucleotide analogs, more preferably, less than about 50 nucleotides ornucleotide analogs, most preferably less than about 35 nucleotide ornucleotide analogs.

[0104] Preferably, the antisense oligonucleotides are complementary tothe 5′ untranslated region of the fetal IGF-II transcript. The“untranslated region of the fetal IGF-II transcript” means that part ofthe IGF-II gene which is transcribed in fetal cells to form the majorIGF-II transcript and which does not form part of the adult IGF-IItranscript (the major transcript in adult cells). Preferably the“untranslated region of the fetal IGF-II transcript” is exons 4, 5 and 6of the IGF-II gene. Most preferably, the “untranslated region of thefetal IGF-II transcript” is that substnatially the sequence of exons 4,5 and 6 as set forth in FIGS. 11 A-C.

[0105] Preferably, the antisense oligonucleotides comprise the sequencesset forth in Tables 1 and 2 (below). TABLE 1 Antisense Sequencesdesigned to target human IGF-II Fetal mRNA SEQ ID ΔG NO. Name Sequence5′-3′ Tm (° C.) (kcal/mol) 1 GT14001 GGC TCG CTG GGG CAG GAG GA 74.6−46.5 2 GT14002 GCT GGT GGG CAG AGC GCG GG 78.0 −48.5 3 GT14003 TTG GTGTCT ACA GCT CAG CA 57.8 −35.2 4 GT14004 CAG CGA GGC AGC GGG CGG CG 82.7−52.5 5 GT14005 TCG GGC GAA GCG GGG ATG GG 79.0 −50.4 6 GT14006 CGG GCCTCG GGA GGG GGA CA 78.2 −49.4 7 GT14007 GAC CGC GGG CGC CCA GCT CG 81.7−51.9 8 GT14008 ACG TCG AGG GGC CGG GGG AG 77.4 −49.3 9 GT14009 CGG GAGAAA GAG CGG GGG CC 75.1 −48.5 10 GT14010 CGA GAG GGC GGG CGT GAG GG 77.0−48.4 11 GT14011 CAG CGA GAG GCG GGC AGG CG 78.2 −49.0 12 GT14012 CGGGCT GTC TTC GGG CTG GG 74.9 −47.0 13 GT14013 GCG ACG GGG CAG AGC GGG GG80.7 −51.4 14 GT14014 CGC TGC CGC CCA CCT CCC TG 77.8 −48.5 15 GT14015TTG GTG TCT GGA AGC CGG CG 72.0 −44.3

[0106] The antisense oligonucleotides were selected from the sequencecomplementary to the human IGF-II mRNA such that the sequence exhibitsthe least likelihood of showing duplex formation, hairpin formation, andhomooligomers/sequence repeats but has a high potential to bind to theIGF-II mRNA sequence and contains a GC clamp. In addition, false primingto other frequently occurring or repetitive sequences in human and mousewas eliminated. These properties were determined using the computermodeling program OLIGO® Primer Analysis Software, Version 5.0(distributed by National Biosciences, Inc., Plymouth, Minn.). TABLE 2Antisense oligonucleotides having a sequence complementary to allregions of the human IGF-II mRNA SEQ ID ΔG NO. Name Sequence 5′-3′ Tm(° C.) (kcal/mol) 16 GT14016 TTC CCC ATT GGG ATT CCC AT 66.8 −42.4 17GT14017 GTC CAC CAG CTC CCC GCC GC 76.9 −47.9 18 GT14018 CGA TGC CAC GGCTGC GAC GG 77.6 −47.6 19 GT14019 ACG CAG GAG GGC AGG CAG GC 74.7 −46.520 GT14020 GCG AGC ACG TGA CCC CGG CG 78.7 −48.6 21 GT14021 CGT GGG CGGGGT CTT GGG TG 75.4 −46.7 22 GT14022 TGT TTC GGG GAG GCG GGG CA 77.5−48.8 23 GT14023 GCG GTA CGA GCG ACG TGC CC 73.8 −45.9 24 GT14024 CAAATG CCG CCG GCC GCA CA 79.7 −49.8 25 GT14025 CGC ATC AGT GCA CGG CCC CC76.5 −46.9 26 GT14026 GTG CGG AAG GCG GCC ACC CT 76.4 −48.2 27 GT14027CAG GGT GCT GAG GGG CGG GC 76.9 −48.0 28 GT14028 GCT CCG GGG CCC AAG CAACC 75.9 −48.3 29 GT14029 CCC TAG GCG CCG CGG TGG TG 77.6 −49.3 30GT14030 TGG CAT GGA CGA CCC CCG GG 77.7 −48.1 31 GT14031 GGG CCG CAA GGTGGA CCG AG 74.8 −46.7

[0107] The antisense oligonucleotides were selected from the sequencecomplementary to the human IGF-II mRNA such that the sequence exhibitsthe least likelihood of showing duplex formation, hairpin formation, andhomooligomers/sequence repeats but has a high potential to bind to theIGF-II mRNA sequence and contains a GC clamp. In addition, false primingto other frequently occurring or repetitive sequences in human and mousewas eliminated. These properties were determined using the computermodeling program OLIGO® Primer Analysis Software, Version 5.0(distributed by National Biosciences, Inc., Plymouth, Minn.).

[0108] In Tables 1 and 2 the “Tm” is the melting temperature of anoligonucleotide duplex calculated according to the nearest-neighbourthermodynamic values. At this temperature 50% of nucleic acid moleculesare in duplex and 50% are denatured. The “AG” is the free energy of theoligonucleotide, which is a measurement of an oligonucleotide duplexstability.

[0109] The term “alky” refers to monovalent alkyl groups preferablyhaving from 1 to 20 carbon atoms and more preferably 1 to 6 carbonatoms. This term is exemplified by groups such as methyl, ethyl,n-propyl, iso-propyl, n-butyl, iso-butyl, n-hexyl, and the like.

[0110] The term “aryl” refers to an unsaturated aromatic carbocyclicgroup of from 6 to 14 carbon atoms having a single ring (e.g., phenyl)or multiple condensed (fused) rings (e.g., naphthyl or anthryl).Preferred aryls include phenyl, naphthyl and the like.

[0111] The term “halo” or “halogen” refers to fluoro, chloro, bromo andiodo and preferably is either fluoro or chloro.

[0112] As to any of the above groups which contain one or moresubstituents, it is understood, of course, that such groups do notcontain any substitution or substitution patterns which are stericallyimpractical and/or synthetically non-feasible. In addition, thecompounds of this invention include all stereochemical isomers arisingfrom the substitution of these compounds.

[0113] The term “pharmaceutically acceptable” means a non-toxic materialthat does not interfere with the effectiveness of the biologicalactivity of the active ingredient(s). The material is compatible with abiological system such as a cell, cell culture, tissue or organism.

[0114] The term “pharmaceutically acceptable salt” refers to salts whichretain the biological effectiveness and properties of the antisenseoligonucleotides of this invention and which are not biologically orotherwise undesirable. In many cases, the antisense oligonucleotides ofthis invention are capable of forming acid and/or base salts by virtueof the presence of amino and/or carboxyl groups or groups similarthereto.

[0115] Pharmaceutically acceptable base addition salts can be preparedfrom inorganic and organic bases. Salts derived from inorganic bases,include by way of example only, sodium, potassium, lithium, ammonium,calcium and magnesium salts. Salts derived from organic bases include,but are not limited to, salts of primary, secondary and tertiary amines,such as alkyl amines, dialkyl amines, trialkyl amines, substituted alkylamines, di(substituted alkyl)amines, tri(substituted alkyl)amines,alkenyl amines, dialkenyl amines, trialkenyl amines, substituted alkenylamines, di(substituted alkenyl)amines, tri(substituted alkenyl)amines,cycloalkyl amines, di(cycloalkyl)amines, tri(cycloalkyl)amines,substituted cycloalkyl amines, disubstituted cycloalkyl amine,trisubstituted cycloalkyl amines, cycloalkenyl amines,di(cycloalkenyl)amines, tri(cycloalkenyl)amines, substitutedcycloalkenyl amines, disubstituted cycloalkenyl amine, trisubstitutedcycloalkenyl amines, aryl amines, diaryl amines, triaryl amines,heteroaryl amines, diheteroaryl amines, triheteroaryl arnines,heterocyclic amines, diheterocyclic amines, triheterocyclic amines,mixed di- and tri-amines where at least two of the substituents on theamine are different and are selected from the group consisting of alkyl,substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl,heterocyclic, and the like. Also included are amines where the two orthree substituents, together with the amino nitrogen, form aheterocyclic or heteroaryl group.

[0116] Examples of suitable amines include, by way of example only,isopropylamine, trimethylamine, diethylamine, tri(iso-propyl)amine,tri(n-propyl)amine, ethanolamine, 2-dimethylaminoethanol, tromethamine,lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline,betaine, ethylenediamine, glucosamine, N-alkylglucamines, theobromine,purines, piperazine, piperidine, morpholine, N-ethylpiperidine, and thelike. It should also be understood that other carboxylic acidderivatives would be useful in the practice of this invention, forexample, carboxylic acid amides, including carboxamides, lower alkylcarboxamides, dialkyl carboxamides, and the like.

[0117] Pharmaceutically acceptable acid addition salts may be preparedfrom inorganic and organic acids. Salts derived from inorganic acidsinclude hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Salts derived from organic acids includeacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,malic acid, malonic acid, succinic acid, maleic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid,salicylic acid, and the like.

[0118] The term “IGF-II gene” or “insulin-like growth factor II” refersto any gene which encodes a protein that is capable of binding to theIGF-I or IGF-II receptor. Preferably the IGF-II gene has one or moreregions with a nucleotide sequence substantially similar to thesequences of exons 4, 5, 6 or 7-9 as set forth in FIGS. 11A-D.

[0119] The term “complementary to” means that the antisenseoligonucleotide sequence is capable of binding to the target sequence,i.e. the IGF-II gene (or mRNA). Preferably, the antisenseoligonucleotide binds to the nucleic acid sequence under physiologicalconditions, e.g. by Watson-Crick base pairing (interaction betweenoligonucleotide and single-stranded nucleic acid) or by Hoogsteen basepairing (interaction between oligonucleotide and double-stranded nucleicacid) or by any other means including in the case of an oligonucleotidebinding to RNA, causing pseudoknot formation. Binding by Watson-Crick orHoogsteen base pairing under physiological conditions is measured as apractical matter by observing interference with the function of thenucleic acid sequence.

[0120] Preferably the antisense oligonucleotide sequence has at leastabout 75% identity with the target sequence, preferably at least about90% identity and most preferably at least about 95% identity with thetarget sequence allowing for gaps or mismatches of several bases.Identity can be determined, for example, by using the BLASTN program ofthe University of Wisconsin Computer Group (GCG) software. Preferablythe antisense oligonucleotide sequence hybridizes to the IGF-II mRNAwith a melting temperature of at least 45° C., more preferably at leastabout 50° C. and most preferably at least about 55° C. as determined bythe OLIGO Primer Analysis Software, version 5.0 program describedherein.

[0121] The term “inhibiting growth” means a reduction or inhibition inthe growth of at least one tumor cell type, preferably by at least 10%,more preferably of at least 50% and most preferably of at least 75%. Theinhibition of growth of tumors can be determined by measuring the sizeof the tumor in nude mice or the inability of the tumor cells to formcolonies in vitro.

[0122] The term “inhibiting metastasis” means reducing or inhibiting thenumber of metastatic tumors that develop, preferably by at least 10% andmore preferably by at least 50%. This can be determined by the methodsset forth in the Examples and other methods known in the art.

[0123] The term “inhibiting expression of IGF-II” means that theantisense oligonucleotide reduces the level of IGF-II mRNA or the levelof IGF-II protein produced by the cell when the oligonucleotide isadministered to the cell.

[0124] The term “mammal” or “mammalian” means all mammals includinghumans, ovines, bovines, equines, swine, canines, felines and mice,etc., preferably it means humans.

[0125] A “mammal suspected of having a tumor” means that the mammal mayhave a proliferative disorder or tumor or has been diagnosed with aproliferative disorder or tumor or has been previously diagnosed with aproliferative disorder or tumor, the tumor has been surgically removedand the mammal is suspected of harboring some residual tumor cells.

[0126] Preparation of the Antisense Oligonucleotides

[0127] The antisense oligonucleotides of the present invention may beprepared by conventional and well-known techniques. For example, theoligonucleotides may be prepared using solid-phase synthesis and inparticular using commercially available equipment such as the equipmentavailable from Applied Biosystems Canada Inc., Mississauga, Canada. Theoligonucleotides may also be prepared by enzymatic digestion of thenaturally occurring IGF-II gene by methods known in the art.

[0128] These oligonucleotides can be prepared by the art recognizedmethods such as phosphoramidate or H-phosphcate chemistry which can becarried out manually or by an automated synthesizer as described byUhlmann et al.(21) and Agrawal et al. (22).

[0129] Isolation and Purification of the Antisense Oligonucleotides

[0130] Isolation and purification of the antisense oligonucleotidesdescribed herein can be effected, if desired, by any suitable separationor purification such as, for example, filtration, extraction,crystallization, column chromatography, thin-layer chromatography,thick-layer chromatography, preparative low or high-pressure liquidchromatography or a combination of these procedures. However, otherequivalent separation or isolation procedures could, of course, also beused.

[0131] An expression vector comprising the antisense oligonucleotidesequence may be constructed having regard to the sequence of theoligonucleotide and using procedures known in the art.

[0132] Vectors can be constructed by those skilled in the art to containall the expression elements required to achieve the desiredtranscription of the antisense oligonucleotide sequences. Therefore, theinvention provides vectors comprising a transcription control sequenceoperatively linked to a sequence which encodes an antisenseoligonucleotide. Suitable transcription and translation elements may bederived from a variety of sources, including bacterial, fungal, viral,mammalian or insect genes. Selection of appropriate elements isdependent on the host cell chosen.

[0133] Reporter genes may be included in the vector. Suitable reportergenes include β-galactosidase (e.g. lacZ), chloramphenicol,acetyl-transferase, firefly luciferase, or an immunoglobulin or portionthereof. Transcription of the antisense oligonucleotide may be monitoredby monitoring for the expression of the reporter gene.

[0134] The vectors can be introduced into cells or tissues by any one ofa variety of known methods within the art. Such methods can be foundgenerally described in Sambrook et al.²⁴; Ausubel et al.²⁵; Chang etal.³⁶; Vega et al.³⁷; and Vectors: A Survey of Molecular Cloning Vectorsand Their Uses³⁸ and include, for example, stable or transienttransfection, lipofection, electroporation and infection withrecombinant viral vectors.

[0135] Introduction of nucleic acids by infection offers severaladvantages. Higher efficiency and specificity for tissue type can beobtained. Viruses typically infect and propagate in specific cell types.Thus, the virus' specificity may be used to target the vector tospecific cell types in vivo or within a tissue or mixed culture ofcells. Viral vectors can also be modified with specific receptors orligands to alter target specificity through receptor mediated events.

[0136] It is contemplated that the oligonucleotide of this invention maybe a ribozyme which cleaves the mRNA. The ribozyme preferably has asequence homologous to a sequence of an oligonucleotide of the inventionand the necessary catalytic center for cleaving the mRNA. For example, ahomologous ribozyme sequence may be selected which destroys the IGF-IImRNA. The ribozyme type utilized in the present invention may beselected from types known in the art. Several ribozyme structuralfamilies have been identified including Group I introns, RNase P, thehepatitis delta virus ribozyme, hammerhead ribozymes and the hairpinribozyme originally derived from the negative strand of the tobaccoringspot virus satellite RNA (sTRSV) (Sullivar 1994, U.S. Pat. No.5,225,347³⁹). Hammerhead and hairpin ribozyme motifs are most commonlyadapted for trans cleavage of mRNAs for gene therapy (Sullivan 1994).Hairpin ribozymes are preferably used in the present invention. Ingeneral, the ribozyme is from 30 to 100 nucleotides in length.

[0137] The oligonucleotides of the invention may be insolubilized. Forexample, the oligonucleotide may be bound to a suitable carrier.Examples of suitable carriers are agarose, cellulose, dextran, Sephadex,Sepharose, carboxymethyl cellulose polystyrene, filter paper,ion-exchange resin, plastic film, plastic tube, glass beads,polyamine-methyl vinyl-ether-maleic acid copolymer, amino acidcopolymer, ethylene-maleic acid copolymer, nylon, silk etc. The carriermay in the shape of, for example, a tube, test plate, beads disc, sphereetc.

[0138] The insoubilized oligonucleotide may be prepared by reacting thematerial with the suitable insoluble carrier using known chemical orphysical methods, for example, cyanogen bromide coupling.

[0139] Pharmaceutical Formulations

[0140] When employed as pharmaceuticals, the antisense oligonucleotidesare usually administered in the form of pharmaceutical compositions.These compounds can be administered by a variety of routes includingoral, rectal, transdermal, subcutaneous, intravenous, intramuscular, andintranasal. These compounds are effective as both injectable and oralcompositions. Such compositions are prepared in a manner well known inthe pharmaceutical art and comprise at least one active compound. Thepharmaceutical composition is, for example, administered intravenously.It is contemplated that the pharmaceutical composition may beadministered directly into the tumor to be treated.

[0141] This invention also includes pharmaceutical compositions whichcontain, as the active ingredient, one or more of the antisenseoligonucleotides associated with pharmaceutically acceptable carriers orexcipients. In making the compositions of this invention, the activeingredient is usually mixed with an excipient, diluted by an excipientor enclosed within such a carrier which can be in the form of a capsule,sachet, paper or other container. When the excipient serves as adiluent, it can be a solid, semi-solid, or liquid material, which actsas a vehicle, carrier or medium for the active ingredient. Thus, thecompositions can be in the form of tablets, pills, powders, lozenges,sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups,aerosols (as a solid or in a liquid medium), ointments containing, forexample, up to 10% by weight of the active compound, soft and hardgelatin capsules, suppositories, sterile injectable solutions, andsterile packaged powders.

[0142] In preparing a formulation, it may be necessary to mill theactive compound to provide the appropriate particle size prior tocombining with the other ingredients. If the active compound issubstantially insoluble, it ordinarily is milled to a particle size ofless than 200 mesh. If the active compound is substantially watersoluble, the particle size is normally adjusted by milling to provide asubstantially uniform distribution in the formulation, e.g. about 40mesh.

[0143] Some examples of suitable excipients include lactose, dextrose,sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate,alginates, tragacanth, gelatin, calcium silicate, microcrystallinecellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, andmethyl cellulose. The formulations can additionally include: lubricatingagents such as talc, magnesium stearate, and mineral oil; wettingagents; emulsifying and suspending agents; preserving agents such asmethyl- and propylhydroxy-benzoates; sweetening agents; and flavoringagents. The compositions of the invention can be formulated so as toprovide quick, sustained or delayed release of the active ingredientafter administration to the patient by employing procedures known in theart.

[0144] The compositions are preferably formulated in a unit dosage form,each dosage containing from about 1% to about 95%, more usually about 5%to about 90% of the active ingredient. The term “unit dosage forms”refers to physically discrete units suitable as unitary dosages forhuman subjects and other mammals, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect, in association with a suitable pharmaceuticalexcipient.

[0145] The antisense oligonucleotide is effective over a wide dosagerange and is generally administered in a pharmaceutically effectiveamount. An effective amount is that amount which when administeredalleviates the symptoms. Preferably the effective amount is that amountable to inhibit tumor cell growth. Preferably the effective amount isfrom about 0.1 mg/kg body weight to about 20 mg/kg body weight. It willbe understood, however, that the amount of the antisense oligonucleotideactually administered will be determined by a physician, in the light ofthe relevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered, theage, weight, and response of the individual patient, the severity of thepatient's symptoms, and the like. The course of therapy may last fromseveral days to several months or until diminution of the disease isachieved. The antisense oligonucleotide may be administered incombination with other known therapies. When co-administered with one ormore other therapies, the oligonucleotide may be administered eithersimultaneously with the other treatments(s), or sequentially. Ifadministered sequentially, the attending physician will decide on theappropriate sequence of administering the oligonucleotide in combinationwith the other therapy.

[0146] For preparing solid compositions such as tablets, the principalactive ingredient/antisense oligonucleotide is mixed with apharmaceutical excipient to form a solid preformulation compositioncontaining a homogeneous mixture of a compound of the present invention.When referring to these preformulation compositions as homogeneous, itis meant that the active ingredient is dispersed evenly throughout thecomposition so that the composition may be readily subdivided intoequally effective unit dosage forms such as tablets, pills and capsules.

[0147] The tablets or pills of the present invention may be coated orotherwise compounded to provide a dosage form affording the advantage ofprolonged action. For example, the tablet or pill can comprise an innerdosage and an outer dosage component, the latter being in the form of anenvelope over the former. The two components can be separated by anenteric layer which serves to resist disintegration in the stomach andpermit the inner component to pass intact into the duodenum or to bedelayed in release. A variety of materials can be used for such entericlayers or coatings, such materials including a number of polymeric acidsand mixtures of polymeric acids with such materials as shellac, cetylalcohol, and cellulose acetate.

[0148] The liquid forms in which the novel compositions of the presentinvention may be incorporated for administration orally or by injectioninclude aqueous solutions, suitably flavored syrups, aqueous or oilsuspensions, and flavored emulsions with edible oils such as corn oil,cottonseed oil, sesame oil, coconut oil, or peanut oil, as well aselixirs and similar pharmaceutical vehicles.

[0149] Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedherein. Preferably the compositions are administered by the oral ornasal respiratory route for local or systemic effect. Compositions inpreferably pharmaceutically acceptable solvents may be nebulized by useof inert gases. Nebulized solutions may be inhaled directly from thenebulizing device or the nebulizing device may be attached to a facemask tent, or intermittent positive pressure breathing machine.Solution, suspension, or powder compositions may be administered,preferably orally or nasally, from devices which deliver the formulationin an appropriate manner.

[0150] The pharmaceutical composition of the invention may be in theform of a liposome, in which the oligonucleotide is combined, inaddition to other pharmaceutically acceptable carriers, with amphipathicagents such as lipids which exist in aggregated form as micells,insoluble monolayers, liquid crystals or lamellar layers which are inaqueous solution. Suitable lipids for liposomal formulation include,without limitation, monoglycerides, diglycerides, sulfatides,lysolecithin, phospholipids, saponin, bile acids and the like. Oneparticularly useful lipid carrier is lipofectin. Preparation of suchliposomal formulations is within the skill in the art, for example,International Patent No. W097/21808 (28) The pharmaceutical compositionmay further include compounds such as cyclodextrins and the like whichenhance delivery of oligonucleotides into cells or slow releasepolymers.

[0151] Another preferred formulation employed in the methods of thepresent invention employs transdermal delivery devices (“patches”). Suchtransdernal patches may be used to provide continuous or discontinuousinfusion of the antisense oligonucleotides of the present invention incontrolled amounts. The construction and use of transdermal patches forthe delivery of pharmaceutical agents is well known in the art. See, forexample, U.S. Pat. 5,023,252⁴⁰, herein incorporated by reference. Suchpatches may be constructed for continuous, pulsatile, or on demanddelivery of pharmaceutical agents.

[0152] Another preferred method of delivery involves “shotgun” deliveryof the naked antisense oligonucleotides across the dermal layer. Thedelivery of “naked” antisense oligonucleotides is well known in the art.See, for example, Felgner et al.,U.S. Pat. No. 5,580,859⁴¹. It iscontemplated that the antisense oligonucleotides may be packaged in alipid vesicle before “shotgun” delivery of the antisenseoligonucleotide.

[0153] The following formulation examples illustrate representativepharmaceutical compositions of the present invention.

FORMULATION EXAMPLE 1

[0154] Hard gelatin capsules containing the following ingredients areprepared: Quantity Ingredient (mg/capsule) Active Ingredient 30.0 Starch305.0 Magnesium stearate 5.0

[0155] The above ingredients are mixed and filled into hard gelatincapsules in 340 mg quantities.

FORMULATION EXAMPLE 2

[0156] A tablet formula is prepared using the ingredients below:Quantity Ingredient (mg/tablet) Active Ingredient 25.0 Cellulose,microcrystalline 200.0 Colloidal silicon dioxide 10.0 Stearic acid 5.0

[0157] The components are blended and compressed to form tablets, eachweighing 240 mg.

FORMULATION EXAMPLE 3

[0158] A dry powder inhaler formulation is prepared containing thefollowing components: Ingredient Weight % Active Ingredient  5 Lactose95

FORMULATION EXAMPLE 4

[0159] Tablets, each containing 30 mg of active ingredient, are preparedas follows: Quantity Ingredient (mg/tablet) Active Ingredient 30.0 mgStarch 45.0 mg Microcrystalline cellulose 35.0 mg Polyvinylpyrrolidone 4.0 mg (as 10% solution in sterile water) Sodium carboxymethyl starch 4.5 mg Magnesium stearate  0.5 mg Talc  1.0 mg Total  120 mg

[0160] The active ingredient, starch and cellulose are passed through aNo. 20 mesh U.S. sieve and mixed thoroughly. The solution ofpolyvinylpyrrolidone is mixed with the resultant powders, which are thenpassed through a 16 mesh U.S. sieve. The granules so produced are driedat 50° to 60° C. and passed through a 16 mesh U.S. sieve. The sodiumcarboxymethyl starch, magnesium stearate, and talc, previously passedthrough a No. 30 mesh U.S. sieve, are then added to the granules which,after mixing, are compressed on a tablet machine to yield tablets eachweighing 120 mg.

FORMULATION EXAMPLE 5

[0161] Capsules, each containing 40 mg of medicament are made asfollows: Quantity Ingredient (mg/capsule) Active Ingredient  40.0 mgStarch 109.0 mg Magnesium stearate  1.0 mg Total 150.0 mg

[0162] The active ingredient, starch, and magnesium stearate areblended, passed through a No. 20 mesh U.S. sieve, and filled into hardgelatin capsules in 150 mg quantities.

FORMULATION EXAMPLE 6

[0163] Suppositories, each containing 25 mg of active ingredient aremade as follows: Ingredient Amount Active Ingredient   25 mg Saturatedfatty acid glycerides to 2,000 mg

[0164] The active ingredient is passed through a No. 60 mesh U.S. sieveand suspended in the saturated fatty acid glycerides previously meltedusing the minimum heat necessary. The mixture is then poured into asuppository mold of nominal 2.0 g capacity and allowed to cool.

FORMULATION EXAMPLE 7

[0165] Suspensions, each containing 50 mg of medicament per 5.0 mL doseare made as follows: Ingredient Amount Active Ingredient 50.0 mg Xanthangum 4.0 mg Sodium carboxymethyl cellulose (11%) Microcrystallinecellulose (89%) 50.0 mg Sucrose 1.75 g Sodium benzoate 10.0 mg Flavorand Color q.v. Purified water to 5.0 mL

[0166] The active ingredient, sucrose and xanthan gum are blended,passed through a No. 10 mesh U.S. sieve, and then mixed with apreviously made solution of the microcrystalline cellulose and sodiumcarboxymethyl cellulose in water. The sodium benzoate, flavor, and colorare diluted with some of the water and added with stirring. Sufficientwater is then added to produce the required volume.

FORMULATION EXAMPLE 8

[0167] Quantity Ingredient (mg/capsule) Active Ingredient  15.0 mgStarch 407.0 mg Magnesium stearate  3.0 mg Total 425.0 mg

[0168] The active ingredient, starch, and magnesium stearate areblended, passed through a No. 20 mesh U.S. sieve, and filled into hardgelatin capsules in 425.0 mg quantities.

FORMULATION EXAMPLE 9

[0169] A formulation may be prepared as follows: A formulation may beprepared as follows: Ingredient Quantity Active Ingredient 5.0 mg CornOil 1.0 mL

FORMULATION EXAMPLE 10

[0170] A topical formulation may be prepared as follows: IngredientQuantity Active Ingredient 1-10 g Emulsifying Wax 30 g Liquid Paraffin20 g White Soft Paraffin to 100 g

[0171] The white soft paraffin is heated until molten. The liquidparaffin and emulsifying wax are incorporated and stirred untildissolved. The active ingredient is added and stirring is continueduntil dispersed. The mixture is then cooled until solid.

[0172] Other suitable formulations for use in the present invention canbe found in Remington's Pharmaceutical Sciences (23).

[0173] The antisense oligonucleotides or the pharmaceutical compositioncomprising the antisense oligonucleotides may be packaged intoconvenient kits providing the necessary materials packaged into suitablecontainers.

[0174] The antisense oligonucleotides of the invention in the form of atherapeutic formulation are useful in treating diseases, and disordersand conditions associated with tumor growth. In such methods atherapeutic amount of a oligonucleotide effective in inhibiting theexpression of fetal transcripts of IGF-II is administered to a cell.This cell may be part of a cell culture, a tissue culture, or may bepart of the whole body of a mammal such as a human.

[0175] The oligonucleotides and ribozymes of the invention modulatetumor cell growth. Therefore methods are provided for interfering orinhibiting tumor cell growth in a mammal comprising contacting the tumoror tumor cells with an antisense oligonucleotide of the presentinvention.

[0176] The term “contact” refers to the addition of an oligonucleotide,ribozyme, etc. to a cell suspension or tissue sample or administeringthe oligonucleotides etc. directly or indirectly to cells or tissueswithin an animal.

[0177] The methods may be used to treat proliferative disordersincluding various forms of cancer or tumors such a leukemias, lymphomas(Hodgkins and non-Hodgkins), sarcomas, melanomas, adenomas, carcinomasof solid tissue, hypoxic tumors, squamous cell carcinomas of the mouth,throat, larynx and lung, genitourinary cancers such as cervical andbladder cancer, hematopoietic cancers, colon cancer, breast cancer,pancreatic cancer, renal cancer, brain cancer, skin cancer, livercancer, head and neck cancers, and nervous system cancers, as well asbenign lesions such as papillomas. Other proliferative disorders such aspsoriasis and those involving arthrosclerosis are also included.

[0178] The oligonucleotides of the invention may also be used to treatdrug resistant tumors. Examples of drug resistant tumors are tumorsresistant to such chemotherapeutic agents as 5-fluorouracil, mitomycinC, methotrexate or hydroxyurea and tumors expressing high levels ofP-glycoprotein which is known to confer resistance to multipleanticancer drugs such as colchicine, vinblastine and doxorubicin; ortumors expressing multi-drug resistance protein as described by Dreeleyet al.(28). Accordingly, it is contemplated that the oligonucleotides ofthe present invention may be administered in conjunction with or inaddition to known anticancer compounds or chemotherapeutic agents.Chemotherapeutic agents are compounds which may inhibit the growth oftumors. Such agents, include, but are not limited to, 5-fluorouracil,mitomycin C, methotrexate and hydroxyurea. It is contemplated that theamount of chemotherapeutic agent may be either an effective amount, i.e.an amount sufficient to inhibit tumor growth or a less than effectiveamount.

[0179] The oligonucleotides of the present invention have been found toreduce the growth of tumors that are metastatic such as C8161 melanomacells. In an embodiment of the invention, a method is provided forreducing the growth of metastastic tumors in a mammal comprisingadministering an effective amount of an oligonucleotide from about 3 toabout 100 nucleotides, comprising a sequence complementary to the 5′untranslated region of mammalian fetal IGF-II mRNA. The sequence may beselected from the group of oligonucleotides shown in Table 1. In anotherembodiment, a method is provided for reducing the growth of metastastictumors in a mammal comprising administering an effective amount of anoligonucleotide from about 20 to about 100 nucleotides, comprising asequence selected from the group of SEQ ID NO: 17-31 set forth in Table2.

[0180] The oligonucleotides may be delivered using viral or non-viralvectors. Sequences may be incorporated into cassettes or constructs suchthat an oligonucleotide of the invention is expressed in a cell.Preferably, the construct contains the proper transcriptional controlregion to allow the oligonucleotide to be transcribed in the cell.

[0181] Therefore, the invention provides vectors comprising atranscription control sequence operatively linked to a sequence whichencodes an oligonucleotide of the invention. The present inventionfurther provides host cells, selected from suitable eucaryotic andprocaryotic cells, which are transformed with these vectors.

[0182] Suitable vectors are known and preferably contain all of theexpression elements necessary to achieve the desired transcription ofthe sequences. Phagemids are a specific example of such beneficialvectors because they can be used either as plasmids or as bacteriophagevectors. Examples of the vectors include viruses such as bacteriophages,baculoviruses, retroviruses, DNA viruses, liposomes and otherrecombination vectors. The vectors can also contain elements for use ineither procaryotic or eucaryotic host systems. One of ordinary skill inthe art will know which host systems are compatible with a particularvector.

[0183] The vectors can be introduced into the cells by stable ortransient transfection, lipofection, electroporation and infection withrecombinant viral vectors.

[0184] Additional features can be added to the vector to ensure itssafety and/or enhance its therapeutic efficacy. Such features include,for example, markers that can be used to negatively select against cellsinfected with recombinant viruses. An example of such a negativeselection marker is the TK gene which confers sensitivity to theantiviral gancyclovir. Features that limit expression to particular celltypes can also be included. Such features include, for example, promoterand regulatory elements that are specific for the desired cell type.

[0185] Retroviral vectors are another example of vectors useful for thein vivo introduction of a desired nucleic acid because they offeradvantages such as lateral infection and targeting specificity. Lateralinfection is the process by which a single infected cell produces manyprogeny virions that infect neighboring cells. The result is that alarge area becomes rapidly infected.

[0186] A vector to be used in the methods of the invention may beselected depending on the desired cell type to be targeted. For example,if breast cancer is to be treated, then a vector specific for epithelialcell may be used. Similarly, if cells of the hematopoietic system are tobe treated, then a viral vector that is specific for blood cells ispreferred.

[0187] Utility

[0188] The antisense oligonucleotides of the present invention may beused for a variety of purposes. They may be used to inhibit theexpression of the IGF-II gene in a mammalian cell, resulting in theinhibition of growth of that cell. The oligonucleotides may be used ashybridization probes to detect the presence of the IGF-II mRNA inmammalian cells. When so used the oligonucleotides may be labeled with asuitable detectable group (such as a radioisotope, a ligand, anothermember of a specific binding pair, for example, biotin). Finally, theoligonucleotides may be used as molecular weight markers.

[0189] In order to further illustrate the present invention andadvantages thereof, the following specific examples are given but arenot meant to limit the scope of the claims in any way.

EXAMPLES

[0190] In the examples below, all temperatures are in degrees Celsius(unless otherwise indicated) and all percentages are weight percentages(also unless otherwise indicated).

[0191] In the examples below, the following abbreviations have thefollowing meanings. If an abbreviation is not defined, it has itsgenerally accepted meaning:

[0192] AS antisense

[0193] cDNA=complementary deoxyribonucleic acid

[0194] ODN oligodeoxynucleotide

[0195] μ=micromolar

[0196] mM millimolar

[0197] M=molar

[0198] ml=milliliter

[0199] μl=microliter

[0200] mg=milligram

[0201] μg=microgram

[0202] PAGE=polyacrylamide gel electrophoresis

[0203] rpm=revolutions per minute

[0204] ΔG=free energy, a measurement of oligonucleotide duplex stability

[0205] kcal=kilocalories

[0206] FBS=fetal bovine serum

[0207] DTT=dithiothrietol

[0208] SDS=sodium dodecyl sulfate

[0209] PBS=phosphate buffered saline

[0210] PMSF=phenylmethylsulfonyl fluoride

[0211] GAPDH=glyceraldehyde-3-phosphate dehydrogenase

[0212] IgG=immunoglobulin G

[0213] kDa=kilodalton

[0214] PCR=polymerase chain reaction

[0215] Tris-HCl=Tris(hydroxymethyl)aminomethane-hydrochloric acid

[0216] TRIzol=total RNA isolation reagent

[0217] ECL=western blotting detection reagents

[0218] IGF-I=insulin-like growth factor I

[0219] IGF-II=insulin-like growth factor II

[0220] UTR=untranslated region

[0221] General Methods in Molecular Biology:

[0222] Standard molecular biology techniques known in the art and notspecifically described were generally followed as in Sambrook et al.²⁴;Ausubel et al.²⁵; and Perbal²⁶.

[0223] Oligonucleotides

[0224] The antisense oligonucleotides were selected from the sequencecomplementary to the IGF-II mRNA such that the sequence exhibits theleast likelihood of showing duplex formation, hairpin formation, andhomooligomers/sequence repeats but has a high potential to bind to theIGF-II mRNA sequence. In addition, a false priming to other frequentlyoccurring or repetitive sequences in human and mouse was eliminated.These properties were determined using the computer modeling programOLIGO® Primer Analysis Software, Version 5.0 International Biosciences,Inc. Plymouth Minn.). Based on this analysis, phosphorothioate antisenseoligonucleotides were designed and then made by methods well known inthe art.

[0225] Cell Lines

[0226] Five different human cancer cell lines including embryonalrhabdomyosarcoma (RD), neuroblastoma (SK-N-AS), Wilms' tumor (G401),melanoma (C8161), human prostate adenocarcinoma (PC-3), metastaticpancreatic adenocarcinoma (AsPC-1) were obtained from American TypeCulture Collection (ATCC). The cell lines were maintained in cc-MEMmedium (Gibco BRL, Gaithersburg, Md.) supplemented with 10% fetal bovineserum (FBS).

Example 1 The Inhibition of Growth of Cancer Cell Lines by AntisenseOligonucleotides Complementary to IGF-II

[0227] The colony forming ability of cancer cell lines treated withdifferent phosphorothioate antisense oligonucleotides was estimatedusing a method previously described (Choy et al.¹⁸). Specifically,aliquots of a tumor cell suspension were seeded into 60 mm tissueculture dishes at a density of approximately 1×10⁴ and incubatedovernight at 37° C. in α-MEM medium supplemented with 10% FBS. Cellswere washed once in 5 ml of PBS and treated with 0.2 μM of the indicatedantisense oligonucleotides in the presence of cationic lipid (Lipofectinreagent, final concentration, 5 μg/ml, Gibco-BRL, Gaithersburg, Md.) for4 hours. The antisense oligonucleotides were removed by washing thecells once with PBS and the cells were cultured in growth medium (α-MEMmedium supplemented with 10% FBS) for 7 to 10 days at 37° C. Colonieswere stained with methylene blue and scored by direct counting asdescribed (Choy et al. 18 and Huang and Wright²⁰). Percent inhibitionwas calculated by comparison with the number of colonies present incultures grown in the absence of antisense oligonucleotides. Allexperiments were performed in quadruplicate.

[0228] The antisense oligonucleotides exerted inhibitory effects on thecolony forming ability of the human tumor cell lines. The percentinhibition of each antisense oligonucleotide is shown in FIG. 2A forrhabdomyosarcoma (RD); FIG. 2B for human prostate cancer cell line(PC-3); FIG. 2C for human pancreatic cancer cell line (AsPC-1); and FIG.2D for human neuroblastoma cell line (SK-N-AS).

Example 2 Decreased mRNA Levels Following Treatment with AntisenseOligonucleotides Complementary to IGF-II

[0229] Human neuroblastoma cells (SK-N-AS) or rhabdomyosarcoma cells(RD) were grown to subconfluency (70-80%) and were treated with 0.2 μMof phosphorothioate antisense oligonucleotides complementary to IGF-IIfor 4 hours in the presence of cationic lipid (Lipofectin reagent, finalconcentration, 5 μg/ml, Gibco-BRL) and Opti-MEM (Gibco-BRL). Cells werewashed once with PBS and incubated for 16 hours in α-MEM medium(Gibco-BRL) containing 10% FBS. Total RNA was prepared in TRIzol reagent(Gibco-BRL) and Northern blot analysis was performed as described inHurta and Wright(27) with some modifications. The blots were hybridizedwith ³²P-labeled 389 bp PCR fragments synthesized using forward primer(5′-TAC CGC CCC AGT GAG ACC CT-3′) [SEQ ID NO:32], reverse primer(5′-TGA CGT TTG GCC TCC CTG AA-3′) [SEQ ID NO:33] and the humancolorectal adenocarcinoma 5′-stretch plus cDNA library (Clonetech, PaloAlto Calif.) as a template. Human IGF-II mRNA was expressed as a ⁻6 kbnucleotide transcript (Werner et al.⁶). Equal RNA loading wasdemonstrated by methylene blue staining of the blot prior tohybridization.

[0230]FIG. 3 shows that the antisense oligonucleotides reduce the IGF-IImRNA levels to at least 50% of the control cells.

Example 3 Decreased IGF-II Protein Levels Following Treatment withAntisense Oligonucleotides Complementary to IGF-II

[0231] Human neuroblastoma cells (SK-N-AS) or rhabdomyosarcoma cells(RD) were grown to subconfluency (70-80%) and were treated with 0.2 μMof phosphorothioate antisense oligonucleotides complementary to IGF-IIfor 4 hours in the presence of cationic lipid (Lipofectin reagent, finalconcentration, 5 μg/ml, Gibco-BRL) and Opti-MEM (Gibco-BRL). Cells werewashed once with PBS and incubated for 20 hours in α-MEM medium(Gibco-BRL) containing 10% FBS. The treatments and incubations wererepeated once more before the whole cell protein extracts were preparedin 2× sample loading buffer (100 mM Tris-HCl, pH 6.8, 0.2 M DTT, 4% SDS,20% glycerol and 0.015% bromophenol blue).

[0232] Western blot analysis was performed as described previously (Choyet al.(18); Fan et al. (19)) with some modification. The expression ofIGF-II was detected with anti-IGF-II antibody (1-2 μg/ml) (ResearchDiagnostics Inc., Flanders N.J.) followed by horseradishperoxidase-conjugated anti-goat IgG (sigma, St. Loius Mo.) at a dilutionof 1:7,000. Approximately 7.5 kDa protein was visualized by ECL(Amersham, Arlington heights, Ill.)

[0233]FIG. 4 shows the reduction in IGF-II protein in humanneuroblastoma cells after treatment with various antisenseoligonucleotides.

[0234]FIG. 5 shows the reduction in IGF-II protein in humanrhabdomyosarcoma cells after treatment with various antisenseoligonucleotides.

Example 4 Inhibition of Human Tumor Cell Growth in Mice by IntravenousTreatment with Antisense Oligonucleotides Complementary to IGF-II

[0235] CD-1 athymic nude mice were purchased from Charles RiverLaboratories (Montreal Canada). SK-N-AS human neuroblastoma cells(typically 3×10⁶ cells in 100 μl of PBS) were subcutaneously injectedinto the right flank of 6-7 weeks old CD-1 athymic female nude mice.Each experimental group included 5 mice. After the size of tumor reachedan approximate volume of 100 mm³, typically 6 days post tumor cellinjection, the various antisense oligonucleotides were administered bybolus infusion into the tail vein every other day at 10 mg/kg. Controlanimals received saline alone for the same period. Treatments typicallylasted 14 days thereafter.

[0236]FIG. 6A shows the effects of the various antisenseoligonucleotides on human neuroblastoma tumor growth in CD-1 nude mice.Antitumor activities were estimated by the inhibition of tumor volume,which was measured with a caliper on average of two day intervals overthe span of 14 days. Each point in the figure represents mean tumorvolume calculated from 5 animals per experimental group. Analysis ofcovariance was used to compare the regression curves of mice over timewithin each treatment group. Specific hypothesis of equality of slopes,or equality of intercepts when slopes are equal are derived from theanalysis. All analysis used the SAS (Statistical Analysis System)version 6.12. When compared to the saline control, administration of theantisense oligonucleotide inhibited the growth of the tumor with a pvalue of <0.0001.

[0237] At the end of the treatment (usually 24 hours after the lasttreatment) the animals were sacrificed and tumor weights were measured.FIG. 6B shows the mean weight of the tumors. The antisenseoligonucleotides showed significant inhibitory effects on tumor growth.One-way analysis of variance was used to compare the means of groups oftreatments. Where the overall group effect was significant, a priorimultiple comparisons using the least square means was used to find thepairs of treatment groups that were significantly different. When tumorweight was compared the antisense oligonucleotides also showedstatistically significant inhibition when compared to the salinecontrol.

Example 5 Inhibition of Human Tumor Cell Growth in Mice by IntravenousTreatment with Antisense Oligonucleotides Complementary to IGF-II

[0238] CD-1 athymic nude mice were purchased from Charles RiverLaboratories (Montreal Canada). G401 human Wilms' tumor cells (typically3×10⁶ cells in 100 μl of PBS) were subcutaneously injected into theright flank of 6-7 weeks old CD-1 athymic female nude mice. Eachexperimental group included 5 mice. After the size of tumor reached anapproximate volume of 100 mm³, typically 8 days post tumor cellinjection, the various antisense oligonucleotides were administered bybolus infusion into the tail vein every other day at 10 mg/kg. Controlanimals received saline alone for the same period. Treatments typicallylasted 18 days thereafter.

[0239]FIG. 7A shows the effects of the various antisenseoligonucleotides on human Wilms' tumor growth in CD-1 nude mice.Antitumor activities were estimated by the inhibition of tumor volume,which was measured with a caliper on average of two day intervals overthe span of 18 days. Each point in the figure represents mean tumorvolume calculated from 5 animals per experimental group. Analysis ofcovariance was used to compare the regression curves of mice over timewithin each treatment group. Specific hypothesis of equality of slopes,or equality of intercepts when slopes are equal are derived from theanalysis. All analysis used the SAS (Statistical Analysis System)version 6.12. When compared to the saline control, administration of theantisense oligonucleotide inhibited the growth of the tumor with a pvalue of ≦0.0002.

[0240] At the end of the treatment (usually 24 hours after the lasttreatment) the animals were sacrificed and tumor weights were measured.FIG. 7B shows the mean weight of the tumors. The antisenseoligonucleotides showed significant inhibitory effects on tumor growth.One-way analysis of variance was used to compare the means of groups oftreatments. Where the overall group effect was significant, a priorimultiple comparisons using the least square means was used to find thepairs of treatment groups that were significantly different When tumorweight was compared the antisense oligonucleotides also showedstatistically significant inhibition when compared to the same control.

Example 6 Reduction in IGF-II mRNA Levels in Human Tumors in Mice byIntravenous Treatment with Antisense Oligonucleotides Complementary toIGF-II

[0241] CD-1 athymic nude mice were purchased from Charles RiverLaboratories (Montreal Canada). SK-N-AS human neuroblastoma cells(typically 3×10⁶ cells in 100 μl of PBS) were subcutaneously injectedinto the right flank of 6-7 weeks old CD-1 athymic female nude mice.Each experimental group included 5 mice. After the size of tumor reachedan approximate volume of 100 mm³, typically 6 days post tumor cellinjection, the various antisense oligonucleotides were administered bybolus infusion into the tail vein every other day at 10 mg/kg. Controlanimals received saline alone for the same period. Mice were sacrificedafter 7 injections and excised tumor fragments of similar size wereimmediately collected into TRIzol reagent (GIBCO BRL) and rapidlyhomogenized for mRNA preparation.

[0242] To measure the effects of antisense oligonucleotides on IGF-IImRNA levels, northern blot analysis was performed as previouslydescribed (Hurta and Wright (27)) with some modifications. The blotswere hybridized with ³²P-labeled 389 bp PCR fragments synthesized usingforward primer (5′-TAC CGC CCC AGT GAG ACC CT-3′) [SEQ ID NO:32],reverse primer (5′-TGA CGT TTG GCC TCC CTG AA-3′) [SEQ ID NO:33] and thehuman colorectal adenocarcinoma 5′-stretch plus cDNA library (Clonetech,Palo Alto Calif.) as a template. Human IGF-II mRNA was expressed as a ⁻6kb nucleotide transcript (Werner et al.⁶) and its levels were comparedto glyceraldehyde-3-phosphate dehydrogenase (GADPH) mRNA as previouslydescribed (Hurta and Wright (27)).

[0243]FIG. 8 shows that the level of IGF-II mRNA was reduced in tumortreated with the antisense oligodeoxynucleotide GTI4006 [SEQ ID NO:6].

Example 7 Reduction in IGF-II Protein Levels in Human Tumors in Mice byIntravenous Treatment with Antisense Oligonucleotides Complementary toIGF-II

[0244] CD-1 athymic nude mice were purchased from Charles RiverLaboratories (Montreal Canada). SK-N-AS human neuroblastoma cells(typically 3×10⁶ (cells in 100 μl of PBS) were subcutaneously injectedinto the right flank of 6-7 weeks old CD-1 athymic female nude mice.Each experimental group included 5 mice. After the size of tumor reachedan approximate volume of 100 mm³, typically 6 days post tumor cellinjection, the various antisense oligonucleotides were administered bybolus infusion into the tail vein every other day at 10 mg/kg. Controlanimals received saline alone for the same period. Mice were sacrificedafter 7 injections and excised tumor fragments of similar size wereimmediately collected into RIPA extraction buffer (50 mM Tris-HCl, pH7.5, 150 mM leupeptin) and rapidly homogenized for protein preparation.

[0245] To measure the effects of antisense oligodeoxynucleotides onIGF-II protein levels, western blot analysis was performed as previouslydescribed (Choy et al. (18), Fan et al. (19)) with some modification.The protein extracts (10-20 μg) were fractionated on a 15% SDS-PAGE geland transferred to nitrocellulose membranes and visualized by India inkstaining. The expression of IGF-II was detected with anti-IGF-IIantibody (1-2 μg/ml) (Research Diagnostics Inc., Flanders N.J.) followedby horseradish peroxidase-conjugated anti-goat IgG (sigma, St. LoiusMo.) at a dilution of 1:7,000. Approximately 7.5 kDa protein wasvisualized by ECL (Amersham, Arlington Heights, Ill.).

[0246]FIG. 9 shows the western blot of the protein extracted from thetumor cells. Each of the antisense oligonucleotides tested reduced theIGF-II protein levels in the tumors. A part of the blot stained withIndia ink is shown underneath to demonstrate an equal loading in eachlane.

Example 8 Inhibition of Experimental Metastasis by AntisenseOligonucleotides

[0247] Experimental metastasis of C8161 human melanoma cells treatedwith different antisense oligonucleotides was estimated as previouslydescribed (Fan et al., 1996¹⁹). Aliquots of cell suspension were seededinto 100 mm tissue culture dishes at a density of 2×10⁶ and incubatedovernight at 37° C. in A-MEM medium supplemented with 10% FBS. Cellswere washed once in 10 ml of PBS and treated with 0.2 μM ofoligonucleotides in the presence of cationic lipid (Lipofectin reagent,final concentration, 5 μg/ml, Gibco-BRL) for 4 hours. The antisenseoligonucleotides were removed by washing the cells once with PBS and thecells were trypsinized. Cells were then collected by centrifugation, andapproximately 1×10⁵ cells suspended in 0.1 ml of PBS were injected intothe tail veins of 6-8 week old CD-1 athymic female nude mice. Estimatesof the number of lung tumors were made 5 weeks later, after excisedlungs from individual mice were stained with picric acid dye solution(75% picric acid, 20% formaldehyde, 5% glacial acetic acid).

[0248]FIG. 10 shows the reduced number of lung tumors in the female nudemice after treatment of the tumor cells with the various antisenseoligodeoxynucleotides.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 37 <210> SEQ ID NO 1<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Human <400> SEQUENCE: 1ggctcgctgg ggcaggagga 20 <210> SEQ ID NO 2 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Human <400> SEQUENCE: 2 gctggtgggc agagcgcggg 20<210> SEQ ID NO 3 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Human<400> SEQUENCE: 3 ttggtgtcta cagctcagca 20 <210> SEQ ID NO 4 <211>LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Human <400> SEQUENCE: 4cagcgaggca gcgggcggcg 20 <210> SEQ ID NO 5 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Human <400> SEQUENCE: 5 tcgggcgaag cggggatggg 20<210> SEQ ID NO 6 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Human<400> SEQUENCE: 6 cgggcctcgg gagggggaca 20 <210> SEQ ID NO 7 <211>LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Human <400> SEQUENCE: 7gaccgcgggc gcccagctcg 20 <210> SEQ ID NO 8 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Human <400> SEQUENCE: 8 acgtcgaggg gccgggggag 20<210> SEQ ID NO 9 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Human<400> SEQUENCE: 9 cgggagaaag agcgggggcc 20 <210> SEQ ID NO 10 <211>LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Human <400> SEQUENCE: 10cgagagggcg ggcgtgaggg 20 <210> SEQ ID NO 11 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Human <400> SEQUENCE: 11 cagcgagagg cgggcaggcg 20<210> SEQ ID NO 12 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Human <400> SEQUENCE: 12 cgggctgtct tcgggctggg 20 <210> SEQ ID NO 13<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Human <400> SEQUENCE:13 gcgacggggc agagcggggg 20 <210> SEQ ID NO 14 <211> LENGTH: 20 <212>TYPE: DNA <213> ORGANISM: Human <400> SEQUENCE: 14 cgctgccgcc cacctccctg20 <210> SEQ ID NO 15 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Human <400> SEQUENCE: 15 ttggtgtctg gaagccggcg 20 <210> SEQ ID NO 16<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Human <400> SEQUENCE:16 ttccccattg ggattcccat 20 <210> SEQ ID NO 17 <211> LENGTH: 20 <212>TYPE: DNA <213> ORGANISM: Human <400> SEQUENCE: 17 gtccaccagc tccccgccgc20 <210> SEQ ID NO 18 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Human <400> SEQUENCE: 18 cgatgccacg gctgcgacgg 20 <210> SEQ ID NO 19<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Human <400> SEQUENCE:19 acgcaggagg gcaggcaggc 20 <210> SEQ ID NO 20 <211> LENGTH: 20 <212>TYPE: DNA <213> ORGANISM: Human <400> SEQUENCE: 20 gcgagcacgt gaccccggcg20 <210> SEQ ID NO 21 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Human <400> SEQUENCE: 21 cgtgggcggg gtcttgggtg 20 <210> SEQ ID NO 22<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Human <400> SEQUENCE:22 tgtttcgggg aggcggggca 20 <210> SEQ ID NO 23 <211> LENGTH: 20 <212>TYPE: DNA <213> ORGANISM: Human <400> SEQUENCE: 23 gcggtacgag cgacgtgccc20 <210> SEQ ID NO 24 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Human <400> SEQUENCE: 24 caaatgccgc cggccgcaca 20 <210> SEQ ID NO 25<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Human <400> SEQUENCE:25 cgcatcagtg cacggccccc 20 <210> SEQ ID NO 26 <211> LENGTH: 20 <212>TYPE: DNA <213> ORGANISM: Human <400> SEQUENCE: 26 gtgcggaagg cggccaccct20 <210> SEQ ID NO 27 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Human <400> SEQUENCE: 27 cagggtgctg aggggcgggc 20 <210> SEQ ID NO 28<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Human <400> SEQUENCE:28 gctccggggc ccaagcaacc 20 <210> SEQ ID NO 29 <211> LENGTH: 20 <212>TYPE: DNA <213> ORGANISM: Human <400> SEQUENCE: 29 ccctaggcgc cgcggtggtg20 <210> SEQ ID NO 30 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Human <400> SEQUENCE: 30 tggcatggac gacccccggg 20 <210> SEQ ID NO 31<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Human <400> SEQUENCE:31 gggccgcaag gtggaccgag 20 <210> SEQ ID NO 32 <211> LENGTH: 20 <212>TYPE: DNA <213> ORGANISM: Human <400> SEQUENCE: 32 taccgcccca gtgagaccct20 <210> SEQ ID NO 33 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Human <400> SEQUENCE: 33 tgacgtttgg cctccctgaa 20 <210> SEQ ID NO 34<211> LENGTH: 419 <212> TYPE: DNA <213> ORGANISM: Human <400> SEQUENCE:34 cccaaaattt gggcattgtt cccgctcgcc ggccacccac tgcagcttcc ccaaccccgc 60gcacagcggg cactggtttc gggcctctct gtctcctacg aagtccccag agcaactcgg 120atttgggaaa tttctctcta gcgttgccca aacacacttg ggtcggccgc gcgccctcag 180gacgtggaca gggagggctt ccccgtgtcc aggaaagcga ccgggcattg cccccagtct 240cccccaaatt tgggcattgt ccccgggtct tccaacggac tgggcgttgc tcccggacac 300tgaggactgg ccccggggtc tcgctcacct tcagcagcgt ccaccgcctg ccacagagcg 360ttcgatcgct cgctgcctga gctcctggtg cgcccgcgga cgcagcctcc agcttcgcg 419<210> SEQ ID NO 35 LENGTH: 1164 <212> TYPE: DNA <213> ORGANISM: Human<400> SEQUENCE: 35 gttcgcctgc tctccggcgg agctgcgtga ggcccggccggccccggccc cccccttccg 60 gccgcccccg cctcctggcc cacgcctgcc cgcgctctgcccaccagcgc ctccatcggg 120 caaggcggcc ccgcgtcgac gccgcccgct gcctcgctgctgactcccgt cccgggcgcc 180 gtccgcgggg tcgcgctccg ccgggcctgc ggattccccgccgcctcctc ttcatctacc 240 tcaactcccc ccatccccgc ttcgcccgag gaggcggttccccccgcagg cagtccggct 300 cgcaggccgc cggcgttgtc accccccccg cgctccccctccagccctcc ccccggcgcg 360 cagcctcggg ccgctcccct ttccgcgctg cgtcccggagcggccccggt gccgccaccg 420 cctgtccccc tcccgaggcc cgggctcgcg acggcagagggctccgtcgg cccaaaccga 480 gctgggcgcc cgcggtccgg gtgcagcctc cactccgccccccagtcacc gcctcccccg 540 gcccctcgac gtggcgccct tccctccgct tctctgtgctccccgcgccc ctcttggcgt 600 ctggccccgg cccccgctct ttctcccgca accttcccttcgctccctcc cgtccccccc 660 agctcctagc ctccgactcc ctccccccct cacgcccgccctctcgcctt cgccgaacca 720 aagtggatta attacacgct ttctgtttct ctccgtgctgttctctcccg ctgtgcgcct 780 gcccgcctct cgctgtcctc tctccccctc gccctctcttcggccccccc ctttcacgtt 840 cactctgtct ctcccactat ctctgccccc ctctatccttgatacaacag ctgacctcat 900 ttcccgatac cttttccccc ccgaaaagta caacatctggcccgccccag cccgaagaca 960 gcccgtcctc cctggacaat cagacgaatt ctccccccccccccaaaaaa aagccatccc 1020 cccgctctgc cccgtcgcac attcggcccc cgcgactcggccagagcggc gctggcagag 1080 gagtgtccgg caggagggcc aacgcccgct gttcggtttgcgacacgcag cagggaggtg 1140 ggcggcagcg tcgccggctt ccag 1164 <210> SEQ IDNO 36 <211> LENGTH: 103 <212> TYPE: DNA <213> ORGANISM: Human <400>SEQUENCE: 36 gcaaactgga tattagcttc tcctgtgaaa gagacttcca gcttcctcctcctcctcttc 60 ctcctcctcc tcctgcccca gcgagccttc tgctgagctg tag 103 <210>SEQ ID NO 37 <211> LENGTH: 4350 <212> TYPE: DNA <213> ORGANISM: Human<400> SEQUENCE: 37 acaccaatgg gaatcccaat ggggaagtcg atgctggtgcttctcacctt cttggccttc 60 gcctcgtgct gcattgctgc ttaccgcccc agtgagaccctgtgcggcgg ggagctggtg 120 gacaccctcc agttcgtctg tggggaccgc ggcttctacttcagcaggcc cgcaagccgt 180 gtgagccgtc gcagccgtgg catcgttgag gagtgctgtttccgcagctg tgacctggcc 240 ctcctggaga cgtactgtgc tacccccgcc aagtccgagagggacgtgtc gacccctccg 300 accgtgcttc cggacaactt ccccagatac cccgtgggcaagttcttcca atatgacacc 360 tggaagcagt ccacccagcg cctgcgcagg ggcctgcctgccctcctgcg tgcccgccgg 420 ggtcacgtgc tcgccaagga gctcgaggcg ttcagggaggccaaacgtca ccgtcccctg 480 attgctctac ccacccaaga ccccgcccac gggggcgcccccccagagat ggccagcaat 540 cggaagtgag caaaactgcc gcaagtctgc agcccggcgccaccatcctg cagcctcctc 600 ctgaccacgg acgtttccat caggttccat cccgaaaatctctcggttcc acgtccccct 660 ggggcttctc ctgacccagt ccccgtgccc cgcctccccgaaacaggcta ctctcctcgg 720 ccccctccat cgggctgagg aagcacagca gcatcttcaaacatgtacaa aatcgattgg 780 ctttaaacac ccttcacata ccctcccccc aaattatccccaattatccc cacacataaa 840 aaatcaaaac attaaactaa cccccttccc ccccccccacaacaaccctc ttaaaactaa 900 ttggcttttt agaaacaccc cacaaaagct cagaaattggctttaaaaaa aacaaccacc 960 aaaaaaaatc aattggctaa aaaaaaaaag tattaaaaacgaattggctg agaaacaatt 1020 ggcaaaataa aggaatttgg cactccccac ccccctctttctcttctccc ttggactttg 1080 agtcaaattg gcctggactt gagtccctga accagcaaagagaaaagaag ggccccagaa 1140 atcacaggtg ggcacgtcgc tcgtaccgcc atctcccttctcacgggaat tttcagggta 1200 aactggccat ccgaaaatag caacaaccca gactggctcctcactccctt ttccatcact 1260 aaaaatcaca gagcagtcag agggacccag taagaccaaaggaggggagg acagagcatg 1320 aaaaccaaaa tccatgcaaa tgaaatgtaa ttggcacgaccctcaccccc aaatcttaca 1380 tctcaattcc catcctaaaa agcactcata ctttatgcatccccgcagct acacacacac 1440 aacacacagc acacgcatga acacagcaca cacacgagcacagcacacac acgagcatac 1500 agcacacaca caaacgcaca gcacacacag cacacagatgagcacacagc acacacacaa 1560 acgcacagca cacacacgca cacacatgca cacacagcacacaaacgcac ggcacacaca 1620 cgcacacaca gtgcacacac agcacacacg caaacgcacacgcacacaca aacgcacagc 1680 acacacgcac acacagcaca cacacgagca cacagcacacaaacgcacag cacacgcaca 1740 cacatgcaca cacagcacac tagcacacag cacacacacaaagacacagc acacacatgc 1800 acacacagca cacacacgcg aacacagcac acacgaacacagcacacaca gcacacacac 1860 aaacacagca cacacatgca cacagcacat gcacacacagcacacacatg aacacagcac 1920 acagcacaca catgcacaca gcacacacgc atgcacagcacacatgaaca cagcacacac 1980 aaacacacag cacacacatg cacacacagc acacacactcatgcgcagca catacatgaa 2040 cacagctcac agcacacaaa cacgcagcac acacgttgcacacgcaagca cccacctgca 2100 cacacacatg cgcacacaca cgcacacccc cacaaaattagatgaaaaca ataagcatat 2160 ctaagcaact acgatatctg tatggatcag gccaaagtcccgctaagatt ctccaatgtt 2220 ttcatggtct gagcccccct cctgttccca tctccactgcccctcggccc tgtctgtgcc 2280 ctgcctctca gaggaggggg ctcagatggt gcggcctgagtgtgcggccg gcggcatttg 2340 ggatacaccc gtaggtgggc ggggtgtgtc ccaggcctaattccatcttt ccaccatgac 2400 agagatgccc ttgtgaggct ggcctccttg gcgcctgtccccacggcccc cgcagcgtga 2460 gccacgatgc tccccatacc ccacccattc ccgatacaccttacttactg tgtgttggcc 2520 cagccagagt gaggaaggag tttggccaca ttggagatggccggtagctg agcagacatg 2580 cccccacgag tagcctgact ccctggtgtg ctcctggaaggaagatcttg gggacccccc 2640 caccggagca cacctaggga tcatctttgc ccgtctcctggggacccccc aagaaatgtg 2700 gagtcctcgg gggccgtgca ctgatgcggg gagtgtgggaagtctggcgg ttggaggggt 2760 gggtgggggg cagtgggggc tgggcggggg gagttctggggtaggaagtg gtcccgggag 2820 attttggatg gaaaagtcag gaggattgac agcagacttgcagaattaca tagagaaatt 2880 aggaaccccc aaatttcatg tcaattgatc tattccccctctttgtttct tggggcattt 2940 ttcctttttt tttttttttt gttttttttt tacccctccttagctttatg cgctcagaaa 3000 ccaaattaaa cccccccccc atgtaacagg ggggcagtgacaaaagcaag aacgcacgaa 3060 gccagcctgg agaccaccac gtcctgcccc ccgccatttatcgccctgat tggattttgt 3120 ttttcatctg tccctgttgc ttgggttgag ttgagggtggagcctcctgg ggggcatggc 3180 catgagcccc cttggagaag tcagagggga gtggagaaggcatgtccggc ctggcttctg 3240 gggacagtgg ctggtcccca gaagtcctga gggcggaggggggggttggg cagggtctcc 3300 tcaggtgtca ggagggtgct cggaggccac aggagggggctcctggctgg cctgaggctg 3360 gccggagggg aaggggctag caggtgtgta aacagagggttccatcagct ggggcagggt 3420 ggccgccttc cgcacacttg aggaaccctc ccctctccctcggtgacatc ttgcccgccc 3480 ctcagcaccc tgccttgtct ccaggaggtc cgaagctctgtgggacctct tgggggcaag 3540 gtggggtgag gccggggagt agggaggtca ggcgggtctgagcccacaga gcaggagagc 3600 tgccaggtct gcccatcgac caggttgctt gggccccggagcccacgggt ctggtgatgc 3660 catagcagcc accaccgcgg cgcctagggc tgcggcagggactcggcctc tgggaggttt 3720 acctcgcccc cacttgtgcc cccagctcag cccccctgcacgcagcccga ctagcagtct 3780 agaggcctga ggcttctggg tcctggtgac ggggctggcatgaccccggg ggtcgtccat 3840 gccagtccgc ctcagtcgca gagggtccct cggcaagcgccctgtgagtg ggccattcgg 3900 aacattggac agaagcccaa agagccaaat tgtcacaattgtggaaccca cattggcctg 3960 agatccaaaa cgcttcgagg caccccaaat tacctgcccattcgtcagga cacccaccca 4020 cccagtgtta tattctgcct cgccggagtg ggtgttcccgggctgcctgt ctgacctccg 4080 tgcctagtcg tggctctcca tcttgtctcc tccccgtgtccccaatgtct tcagtggggg 4140 gccccctctt gggtcccctc ctctgccatc acctgaagacccccacgcca aacactgaat 4200 gtcacctgtg cctgccgcct cggtccacct tgcggcccgtgtttgactca actcagctcc 4260 tttaacgcta atatttccgg caaaatccca tgcttgggttttgtctttaa ccttgtaacg 4320 cttgcaatcc caataaagca ttaaaagtca 4350

What is claimed:
 1. A method for inhibiting the growth of a human tumorcomprising, administering to a human suspected of having the tumor aneffective amount of an antisense oligonucleotide comprising from about20 to 100 nucleotides comprising a sequence selected from the groupconsisting of SEQ ID NOs:17-31 in Table 2 under conditions such that thegrowth of the tumor is inhibited.
 2. The method according to claim 1wherein the oligonucleotide is nuclease resistant.
 3. The methodaccording to claim 1 further comprising the step of administering to thehuman a chemotherapeutic agent.
 4. A method for inhibiting themetastasis of a human tumor comprising, administering to a humansuspected of having a metastatic tumor an effective amount of anantisense oligonucleotide from about 20 to 100 nucleotides comprising asequence complementary to the 5′ untranslated region consisting of exons4, 5 or 6 of human fetal IGF-II mRNA under conditions such thatmetastasis of the tumor is inhibited.
 5. The method according to claim 4further comprising the step of administering to the human achemotherapeutic agent.
 6. The method according to claim 4 wherein theoligonucleotide is nuclease resistant.
 7. The method according to claim4 wherein the oligonucleotide comprises a sequence selected from thegroup consisting of SEQ ID NOs:1-15.
 8. A method for inhibiting themetastasis of a human tumor comprising, administering to a humansuspected of having a metastatic tumor an effective amount of anantisense oligonucleotide from about 20 to 100 nucleotides comprising asequence selected from the group consisting of SEQ ID NOs:17-31 underconditions such that metastasis of the tumor is inhibited.
 9. The methodaccording to claim 8 further comprising the step of administering to thehuman a chemotherapeutic agent.
 10. The method according to claim 9wherein the oligonucleotide is nuclease resistant.
 11. The methodaccording to claim 4, wherein the antisense oligonucleotide comprisesfrom about 20 to about 50 nucleotides.